Regulation of codBAOperon Expression in Escherichia coliby UTP-dependent Reiterative Transcription and UTP-sensitive Transcriptional Start Site Switching

Abstract

Reiterative transcription is the repetitive addition of nucleotides to the 3′ end of a nascent transcript due to slippage between the transcript and DNA template. Recently, we showed that pyrimidine-mediated regulation of pyrBIoperon expression in Escherichia colioccurs, in part, through a mechanism in which induction of UTP-dependent reiterative transcription within the initially transcribed region prevents downstream extension of the nascent transcript to include structural gene sequences. In this study we demonstrate that pyrimidine-mediated regulation of codBAoperon expression in E. colialso involves UTP-dependent reiterative transcription during initiation; however, the mechanism is different from that of the pyrBIoperon. The initially transcribed region of the codBApromoter contains the sequence GATTTTTTG (non-template strand). Our results show that transcription is initiated primarily at the first two bases designated G7 and A8 (counting from the −10 region). When transcripts are initiated at position A8, UTP-dependent reiterative transcription always occurs within the run of T residues in the initially transcribed region. The AUUUU n (where n=1 to >15) transcripts produced by this reaction are not extended productively to include downstream codBAsequences. In contrast, most transcripts initiated at position G7 do not engage in reiterative transcription and can be elongated normally. Characterization of a codBApromoter mutation that prevents reiterative transcription showed that this reaction is required for virtually all pyrimidine-mediated regulation of operon expression and that UTP levels control the selection of the G7 and A8 transcriptional start sites. These results suggest a model for regulation in which high intracellular levels of UTP favor transcriptional initiation at position A8 and thus the accompanying reiterative transcription, which together preclude initiation at position G7. Low levels of UTP inhibit initiation at position A8 and the associated reiterative transcription, thereby allowing high levels of initiation at position G7 and operon expression. Our results also indicate critical sequence requirements for reiterative transcription, which are important for understanding the mechanism of this reaction as well as for identifying other promoters at which this reaction may occur. Of particular interest is the indication that an RNA : DNA hybrid forms during transcriptional initiation and the strength of this hybrid controls the extent of reiterative transcription.